ABSTRACT Ourcurrentbedsidetestsforurinarytractinfections(UTI)arenotsufficientlyaccurate.Thebestavailabletest forUTI,theleukocyteesterase(LE)test,hasasensitivityofonly79%,meaningthat21%ofchildrenwitha trueUTIaremissed,placingthematincreasedriskforpermanentrenalscarring.The87%specificityoftheLE testmeansthat13%ofthosewithoutUTIwillbeincorrectlydiagnosedashavingUTIandreceiveantimicrobial treatment.Accordingly,developmentofmoresensitiveandspecifictestsforthediagnosisofUTI,whichisour firstaim,representsanimportantcontribution.Wewillcollecturinespecimensfrom3,334consecutivefebrile childrenaged1monthto3yearswithpresumedUTIand,inasampleofcasesandcontrolsfromthislarger population,wewillperformtranscriptomics(mRNA)andmultiplexinflammatoryproteinanalysistoidentify urinarymarkersthatdistinguishchildrenwithUTIfromfebrilechildrenwithoutUTI.Promisingmarkerswillbe validatedinasampleselectedfrom3,334childrenfromasecondsite.Asecondlimitationinthecurrentclinical approachtoUTIstemsfromthelackofreliablemarkerstodistinguishchildrenwithbladderinfectionfrom childrenwithkidneyinfection(pyelonephritis).Becauseaccuratemarkersforpyelonephritishavenotbeen identified,allfebrilechildrenwithUTIaregenerallyassumedtohavepyelonephritis(i.e.,treatedwithantibiotics for10daysandundergorenalultrasonography)eventhoughapproximatelyonly40%havepyelonephritis.To identifyaccuratemarkersforpyelonephritis,whichisoursecondaim,wewillenroll100febrilechildrenwith UTIandperformtranscriptomicsandmultiplexinflammatoryproteinanalysisontheirurinesamples. PyelonephritiswillbeconfirmedusingTc99m-dimercaptosuccinicacid(DMSA)scan.Wewillvalidate promisingmarkersinanindependentsampleof100childrenfromasecondsite.DiagnosisofUTIisespecially challenginginchildrenwithinfectionscausedbybacteriaotherthanEscherichiacoli.Accordingly,ourthirdaim willbetodeveloptestsforthediagnosisofUTIinthesechildren.Featuresthatwilldifferentiateourstudy include(1)enrollmentofconsecutivefebrilesubjects(whichwillminimizebias);?(2)useofcatheterizationto collectallurinesamples(whichwillminimizemisclassification);?(3)timelyprocessingoftheurinesamples,(4) useofanovelmethodofassemblinghigh-qualityRNAlibrariesfromsmallamountsofurine,and(5)selection ofdifferentsitesfortrainingandvalidation.Resultsfromtheproposedprojectareexpectedto(1)identifyeasy tomeasureurinaryproteinsthatcanbeusedtoaccuratelydiagnoseUTIinchildrenwithandwithoutE.coli infection,(2)developRNAsignaturesforUTIandpyelonephritiswhichcanbeusedasagoldstandardin futurestudies,and(3)establishalargespecimenbankforuseinfuturestudies.